Gö-VIP- 23: Knut Brockmann, Simone Schröder

Name der Einrichtung: Sozialpädiatrisches Zentrum, Klinik für Kinder- und Jugendmedizin, UMG

Titel der Publikation: Heterozygous truncating variants in SUFU cause congenital ocular motor

In: Genetics in Medicine, 2020 Oct 7. doi: 10.1038/s41436-020-00979-w. Epub ahead of print. PMID: 33024317.

Autoren: Simone Schröder1*, Yun Li2*, Gökhan Yigit2*, Janine Altmüller3, Ingrid Bader4, Andrea Bevot5, Saskia Biskup6, Steffi Dreha-Kulaczewski1, G. Christoph Korenke7, Raimund Kottke8, Johannes A. Mayr9, Martin Preisel9, Sandra P. Toelle10, Sarah Wente-Schulz11, Saskia B. Wortmann9,12, Heidi Hahn2, Eugen Boltshauser10, Anja Uhmann2, Bernd Wollnik2,13, and Knut Brockmann1#

1 Interdisciplinary Pediatric Center for Children with Developmental Disabilities and Severe Chronic Disorders, Department of Pediatrics and Adolescent Medicine, University Medical Center, Göttingen, D-37075, Germany. 2 Institute of Human Genetics, University Medical Center, Göttingen, D-37075, Germany. 3 Cologne Center for Genomics, Center for Molecular Medicine Cologne, University of Cologne, Cologne, D-50931, Germany. 4 Department of Clinical Genetics, University Children's Hospital, Paracelsus Medical University, Salzburg, A-5020, Austria. 5 Department of Pediatric Neurology, University Hospital Tübingen, Tübingen, D-72076, Germany. 6 Praxis für Humangenetik Tübingen, Tübingen, D-72076, Germany. 7 Department of Pediatric Neurology, University Hospital Oldenburg, Oldenburg, D-26133, Germany. 8 Department of Diagnostic Imaging, University Children’s Hospital, Zurich, CH-8032, Switzerland. 9 Department of Pediatrics, University Hospital Salzburg, Paracelsus Medical University, Salzburg, A-5020, Austria. 10 Department of Pediatric Neurology, University Children’s Hospital, Zurich, CH-8032, Switzerland. 11 Department of Pediatric , Liver and Metabolic Diseases, Hannover Medical School Children's Hospital, Hannover, D-30625, Germany. 12 Radboud Center for Mitochondrial Medicine, Department of Pediatrics, Amalia Children’s Hospital, Radboudumc, Nijmegen, NL-6500, The Netherlands. 13 Cluster of Excellence "Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells" (MBExC), University of Göttingen, Göttingen, D-37075, Germany.

#Corresponding Author * These authors contributed equally to this work.

Zusammenfassung des wissenschaftlichen Inhalts

(Knut Brockmann, Simone Schröder)

Der an der Harvard Medical School tätige amerikanische Augenarzt David G. Cogan prägte im Jahre 1952 den Begriff „congenital ocular motor apraxia“ (COMA), als er in einer Jackson Memorial Lecture vier Kinder beschrieb, die nicht in der Lage waren, Sakkaden, also willkürliche rasche Blickzielbewegungen auszuführen. In seiner Originalpublikation beschrieb er COMA als Symptom, nicht als Entität.

Da in den Jahren nach dieser klinischen Erstbeschreibung COMA überwiegend bei Patienten berichtet wurde, die zugleich eine frühmanifeste Ataxie und allgemeine Entwicklungsverzögerung aufwiesen, z. T. auch familiär, entwickelte sich ein Konzept von COMA als Krankheitsentität mit genetischer Grundlage und gemutmaßter autosomal- rezessiver Vererbung. Ein assoziiertes Gen konnte jedoch nie gefunden werden.

Diese Position der COMA als isolierte klinische Entität wurde jedoch zunehmend in Zweifel gezogen, da frühmanifeste (kongenitale) okuläre Apraxie bei einer Vielzahl seltener neurogenetischer Erkrankungen, bei speziellen Hirnfehlbildungen, aber auch bei erworbenen neurologischen Erkrankungen auftreten kann. Besonders häufig ist COMA als klinisches Merkmal des Joubert-Syndroms erkannt worden, einer wiederum genetisch heterogenen Ziliopathie. Bei Patienten mit Joubert-Syndrom liegt als pathognomonischer Befund in der MR- Tomographie des Hirns das sog. „Molar Tooth Sign“ vor, das auf eine Fehlbildung der Kleinhirnschenkel und des Mittelhirns zurückzuführen ist.

Mit dem Ziel, das klinische und neuroradiologische Spektrum der COMA genauer zu charakterisieren und die genetischen Grundlagen aufzuklären, rekrutierten wir eine Kohorte von 15 Patienten aus 6 Familien mit COMA, bei denen nach Anamnese, klinischem Befund und MRT keine definitive diagnostische Zuordnung zu einem der bekannten mit COMA assoziierten Krankheitsbilder möglich war, insbesondere kein Joubert-Syndrom mit Molar Tooth Sign vorlag.

Mittels Exom-Sequenzierung gelang es Dr. Yun Li aus der Arbeitsgruppe von Prof. Wollnik, Institut für Humangenetik der UMG, zunächst bei 3 Patienten, „Suppressor of Fused (SUFU)“ als Kandidaten-Gen zu identifizieren. In Kooperation mit Kollegen aus Neuropädiatrie und Humangenetik in Oldenburg, Tübingen, Zürich und Salzburg konnten wir insgesamt 6 neue heterozygote krankheitsverursachende Varianten im SUFU Gen als Ursache der COMA in diesen Familien nachweisen und diese Varianten näher charakterisieren.

SUFU ist ein negativer Regulator des Hedgehog-Signalweges. Funktionelle Studien an Patienten-Fibroblasten zeigten keine Unterschiede im Zilienvorkommen, Morphologie oder Lokalisierung von Zilienproteinen. Allerdings zeigte die Expressionsanalyse von Hedgehog- Zielgenen eine signifikante Zunahme der allgemeinen Signalaktivität.

Nochmalige Re-Evaluation der MRT-Bilder ergab wiederum kein voll ausgeprägtes Molar Tooth Sign, aber doch diskrete morphologische Kleinhirn-Anomalien. Der klinische Phänotyp und diese MRT-Merkmale sprechen zusammengenommen dafür, dass es sich bei der mit heterozygoten trunkierenden SUFU-Varianten assoziierten COMA um eine von uns erstmals beschriebene forme fruste des Joubert-Syndroms handelt. Weitere Informationen:

Prof. Knut Brockmann Universitätsmedizin Göttingen Sozialpädiatrisches Zentrum, Klinik für Kinder- und Jugendmedizin Robert-Koch-Str. 40, 37075 Göttingen Tel. 0551-39-10358 (Sekretariat),, 39-66299 (Büro) Email: [email protected]

ARTICLE

Heterozygous truncating variants in SUFU cause congenital ocular motor apraxia

Simone Schröder, PhD1, Yun Li, PhD2, Gökhan Yigit, PhD2, Janine Altmüller, MD3, Ingrid Bader, MD, MSc4, Andrea Bevot, MD5, Saskia Biskup, MD,PhD6, Steffi Dreha-Kulaczewski, MD1, G. Christoph Korenke, MD7, Raimund Kottke, MD8, Johannes A. Mayr, MD9, Martin Preisel, MD9, Sandra P. Toelle, MD10, Sarah Wente-Schulz, MD11, Saskia B. Wortmann, MD9,12, Heidi Hahn, MD2, Eugen Boltshauser, MD10, Anja Uhmann, PhD2, Bernd Wollnik, MD2,13 and Knut Brockmann, MD 1

Purpose: This study aimed to delineate the genetic basis of target genes detected a significant increase in the general signaling congenital ocular motor apraxia (COMA) in patients not otherwise activity in COMA patient–derived fibroblasts compared with classifiable. control cells. We observed higher basal HH signaling activity resulting in increased basal expression levels of GLI1, GLI2, GLI3, Methods: We compiled clinical and neuroimaging data of Patched1 individuals from six unrelated families with distinct clinical features and . Neuroimaging revealed subtle cerebellar changes, but of COMA who do not share common diagnostic characteristics of no full-blown molar tooth sign. or other known genetic conditions associated Conclusion: Taken together, our data imply that the clinical with COMA. We used exome sequencing to identify pathogenic phenotype associated with heterozygous truncating germline variants and functional studies in patient-derived fibroblasts. variants in SUFU is a forme fruste of Joubert syndrome. Results: In 15 individuals, we detected familial as well as de novo heterozygous truncating causative variants in the Suppressor of Genetics in Medicine (2020) https://doi.org/10.1038/s41436-020- Fused (SUFU) gene, a negative regulator of the Hedgehog (HH) 00979-w signaling pathway. Functional studies showed no differences in cilia occurrence, morphology, or localization of ciliary proteins, such as Keywords: SUFU; congenital ocular motor apraxia; COMA; smoothened. However, analysis of expression of HH signaling sonic hedgehog; Joubert syndrome

– INTRODUCTION and global developmental delay was emphasized.3 9 Most of The term congenital ocular motor apraxia (COMA), intro- the patients described experienced gradual resolution of OMA duced by Cogan in 1952, designates the inability to initiate and ataxia over their first decade of life, whereas cognitive – saccades, i.e., the eye movements performing rapid gaze shift. impairment persisted to a variable extent.4,7 9 These reports In his original report, Cogan described four children with a shaped a concept of COMA as a clinical entity and likely distinct disturbance of voluntary horizontal gaze character- inherited as an autosomal recessive disorder, although Cogan, ized by the “inability to turn the eyes voluntarily in a direction in his original report, described COMA as a symptom, not a for which there is full involuntary…control” accompanied by diagnosis. However, no gene associated with isolated COMA compensatory, jerky head movements.1 COMA usually affects (OMIM 257550) has been identified yet. horizontal, but rarely also vertical saccades. Of note, this condition is sometimes also called Cogan Ocular motor apraxia (OMA) is observed in a wide range of syndrome type 2, thus distinguishing it from Cogan syndrome conditions.2 A frequent and consistent co-occurrence of early- type 1, a rare inflammatory disorder characterized by onset (congenital) OMA, also designated infantile-onset interstitial keratitis and sensorineural hearing loss, occasion- saccade initiation delay, with early-onset cerebellar ataxia ally accompanied by systemic vasculitis.

1Interdisciplinary Pediatric Center for Children with Developmental Disabilities and Severe Chronic Disorders, Department of Pediatrics and Adolescent Medicine, University Medical Center, Göttingen, Germany; 2Institute of Human Genetics, University Medical Center, Göttingen, Germany; 3Cologne Center for Genomics, Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany; 4Department of Clinical Genetics, University Children’s Hospital, Paracelsus Medical University, Salzburg, Austria; 5Department of Pediatric Neurology, University Hospital Tübingen, Tübingen, Germany; 6Praxis für Humangenetik Tübingen, Tübingen, Germany; 7Department of Pediatric Neurology, University Hospital Oldenburg, Oldenburg, Germany; 8Department of Diagnostic Imaging, University Children’s Hospital, Zurich, Switzerland; 9Department of Pediatrics, University Hospital Salzburg, Paracelsus Medical University, Salzburg, Austria; 10Department of Pediatric Neurology, University Children’s Hospital, Zurich, Switzerland; 11Department of Pediatric Kidney, Liver and Metabolic Diseases, Hannover Medical School Children’s Hospital, Hannover, Germany; 12Radboud Center for Mitochondrial Medicine, Department of Pediatrics, Amalia Children’s Hospital, Radboudumc, Nijmegen, The Netherlands; 13Cluster of Excellence “Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells” (MBExC), University of Göttingen, Göttingen, Germany. Correspondence: Knut Brockmann ([email protected]) These authors contributed equally: Simone Schröder, Yun Li, Gökhan Yigit. Submitted 24 June 2020; revised 11 September 2020; accepted: 17 September 2020

GENETICS in MEDICINE | Volume 0 | Number 0 | Month 1 ARTICLE SCHRÖDER et al

More recently, doubts have arisen “whether Cogan-type Clinical and qualitative neuroimaging analysis oculomotor apraxia can exist as an isolated entity.”10 Liu and Data on developmental course and neurological features were coworkers11 assigned COMA to three major clinical condi- collected by review of the clinical histories and by tions: (1) benign or idiopathic variant with normal neuroima- clinical–neurological follow-up investigations. Information ging and only occasionally occurring neurologic symptoms; about ophthalmological features was compiled from neuro- (2) nonprogressive, noninherited variant with structural brain ophthalmological or pediatric neurological reports. Cognitive anomaly caused, e.g., by dysgenesis of the cerebellar vermis or function was assessed using standardized neuropsychological corpus callosum, inferior vermian hypoplasia, Dandy–Walker tests whenever possible, or it was appraised from the patient’s malformation, gray matter heterotopias, and perinatal ische- history, clinical examination, and school reports. Additional mia; and (3) “part of a genetic syndrome” variant, which information was gathered in telephone interviews with the includes, e.g., Joubert syndrome, Jeune syndrome, and a patients or their parents using a standardized questionnaire. subset of patients with Leber congenital amaurosis. Neuroimaging data were available for ten subjects. Two In a previous study aimed at a nosological delineation of subjects received a new MRI in line with this study for COMA, we investigated a cohort of 21 patients diagnosed as technically optimized assessment of the hindbrain. All having COMA.12 In that study, a reappraisal of neuroimaging available MRI data sets were qualitatively analyzed by two revealed a molar tooth sign (MTS) indicative of Joubert pediatric neurologists with experience in neuroimaging of the syndrome in 11 and specific neuroimaging features pointing brainstem and cerebellum. All imaging sequences in axial, to other diagnoses in 2 patients. The remaining eight subjects coronal, and sagittal orientation were scrutinized with a focus had normal magnetic resonance image (MRI) or nonconclu- on size and position of the superior cerebellar peduncles, sive neuroimaging features, which led to a descriptive hypo-/dysplasia of the cerebellar vermis, cerebellar , diagnostic classification of COMA.12 brainstem morphology including shape of the interpeduncu-

1234567890():,; Pursued investigation of these eight patients and recruit- lar fossa at the section of the brainstem isthmus and upper ment of further subjects with COMA framed the study pons, size and shape of the 4th ventricle, and any other presented here. We report six unrelated families comprising cerebellar or cerebral malformations, as described 15 affected individuals with distinct clinical features of previously.12 COMA who do not share common diagnostic characteristics of Joubert syndrome or other known genetic conditions. We Exome sequencing and variant screening identified heterozygous truncating germline variants in the In families 1, 2, 3, and 6,13 trio-based exome sequencing (ES) Suppressor of Fused (SUFU) gene and were able to show de was performed. In family 4, a next-generation sequencing novo occurrence of these SUFU variants in two families and (NGS) panel of 13 genes associated with was inheritance from a mildly affected parent in the remaining applied. All detected SUFU variants were confirmed by four families. On a cellular level, COMA patient–derived polymerase chain reaction (PCR) amplification and subse- fibroblasts show a generally high basal Hedgehog (HH) quent Sanger sequencing on an independent DNA sample signaling activity, which, however, can be regulated by and tested for cosegregation within the respective families. In exogenous HH signaling activator or inhibitor treatment. the affected subject II.1 of family 5, all exons and adjacent exon–intron boundaries of SUFU were analyzed by PCR and MATERIALS AND METHODS subsequent, bidirectional Sanger sequencing in a candidate Subjects gene approach. We compiled clinical data of 15 subjects from six unrelated Details of ES and variant screening as well as methods of families. Clinical phenotypes and MRI data of three subjects cell culture and treatments, analysis of cilia formation, were included in our previous report.12 An additional six immunofluorescence staining, real-time quantitative PCR, subjects with heterozygous SUFU variants and, in most cases, and statistical analysis are provided as Supplementary early-onset ocular apraxia were recruited from the families of Information. these three index patients. Six further subjects, five of them with COMA, from three families were recruited through RESULTS national and international collaborations with the attending Clinical and neuroimaging phenotypes of individuals with neurologists. COMA Through national and international collaborations, we Ethics statement recruited 15 individuals from six families with a clinical All studies were performed in accordance with the Declara- diagnosis of COMA and without conclusive neuroimaging tion of Helsinki protocols. The studies were reviewed and features, notably without definite molar tooth sign. Three of approved by the local institutional ethics board (University these patients, III.6 (family 1), II.1 (family 2), and II.1 (family Medical Center Göttingen, Göttingen, Germany; file numbers 3), were presented in a previous clinical study without any 19/5/14 and 3/9/14). Written informed consent was obtained genetic findings.12 Detailed clinical information of affected from all affected subjects, parents, or legal representatives individuals is summarized in Table 1. Brain MRI was available participating in this study. from ten subjects including four from a parent, and was

2 Volume 0 | Number 0 | Month | GENETICS in MEDICINE EEISi MEDICINE in GENETICS SCHRÖDER Table 1 Clinical and genetic features of 15 individuals with heterozygous SUFU variants. Family Patient Sex Current SUFU variant Development Neurological findings MRI of

# #(#in age the brain al et (origin) Wente (years) available et al.12) cDNA position AA change Unaided Speech delay Ocular Early- Cognitive Head walking at apraxia onset ataxia development circumference age (months) (onset, course) at last follow-

| up (SD) oue0 Volume 1 (T) I:2 F 64 c.83C>A p.Ser28* 11 Yes No n.a. n.a. - 0.6 No II:3 F 41 c.83C>A p.Ser28* “Normal” Yes No No Learning - 0.2 No

| disability ubr0 Number II:5 F 37 c.83C>A p.Ser28* 10 No No No Normal + 0.2 No II:8 M 21 c.83C>A p.Ser28* 32 Yes 5 months, ↓ Yes Learning + 1.3 Yes disability III:3 M 6 c.83C>A p.Ser28* 20 Yes 5 months, ↓ Yes Delayed + 1.0 No | Month III:6 (10) M 13 c.83C>A p.Ser28* 24 Yes 6 months, ↓ Yes Learning + 0.1 Yes disability 2 (S/ I:1 M 42 c.1099G>T p.Glu367* 27 No No Yes Normal + 1.3 Yes CH) II:1 (14) M 11 c.1099G>T p.Glu367* 24 Yes 6 months, ↓ Yes Normal + 0.9 Yes 3 (D) II:1 (19) F 10 c.479delA p.His160Leufs*20 14 No 6 months, ↓ No Normal + 2.6 Yes (de novo) 4 (AL) II:1 M 2 c.1220_1221ins p.Phe408Valfs*13 21 No 8 months, ↓ Yes Normal - 0.2 Yes T (de novo) 5 (CH) I:1 M 40 c.309_310delAG p.Arg103Serfs*3 “Late” No Early No Normal + 3.0 Yes childhood, still present II:1 M 6 c.309_310delAG p.Arg103Serfs*3 22 Yes 6 months, ↓ Yes Normal + 0.6 Yes 6 (A) I:2 F 37 c.[1333dupG] p.[Glu445Glyfs*22] 24 No No No Normal + 1.3 Yes II:1 M 5 c.[1333dupG] p.[Glu445Glyfs*22] 19 No 6 months, ↓ Yes Normal + 1.8 Yes II:2 M 2 c.[1333dupG] p.[Glu445Glyfs*22] 21 Yes 10 months, = Yes n. a. + 2.1 No A Austrian origin, AA amino acid, AL Albanian origin, cDNA complementary DNA, CH Swiss origin, D German origin, MRI magnetic resonance image, MTS molar tooth sign, n. a. not available, OA ocular apraxia, S Sardi- nian origin, SCP superior cerebellar peduncles, SD standard deviation, T Turkish origin, ↓ attenuating, = unchanged. ARTICLE 3 ARTICLE SCHRÖDER et al

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ij k l

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Fig. 1 Characteristic magnetic resonance image (MRI) features in three subjects with heterozygous SUFU variants. Four representative T1- weighted MRIs (arranged in horizontal rows) are shown from three individuals with SUFU variants and one adult healthy control (m–p). Panels (a–d) are from subject I.1, family 3, at age 1.5 years; (e–h) from subject II.1, family 2, at age 7.5 years; and (i–l) from subject II.1, family 5, at age 40 years. The first vertical row (a,e,i,m) shows axial views at the level of the upper vermis, indicating folial dysplasia (arrow). The second vertical row (b,f,j,n) illustrates axial views at the level of the superior cerebellar peduncles (arrows), these are more prominent (longer, thicker) compared with normal. The third vertical row (c,g,k,o) shows parasagittal sections demonstrating that the superior cerebellar peduncles (arrows) are thicker and have a more horizontal course compared with normal (o). The fourth vertical row (d,h,l,p) illustrates coronal images revealing irregular folia and vermis splitting (arrows) in the individuals with SUFU variants. deemed conspicuous in families 3 through 6 already at first observe any signs of a classical, full-blown molar tooth sign. evaluation. Standardized qualitative reanalysis of all MRI data However, the superior cerebellar peduncles were abnormally sets revealed strikingly similar neuroimaging features in all prominent in all cases, thickened, elongated, and had a more subjects. Figure 1 illustrates the relevant findings from three horizontal course, best seen in parasagittal sections. On axial individuals in comparison with a healthy control. We did not sections through the upper vermis, a cerebellar folia dysplasia

4 Volume 0 | Number 0 | Month | GENETICS in MEDICINE SCHRÖDER et al ARTICLE was obvious in eight cases and present in a milder form in two normal. She had surgical excision of a digital tumor at age six individuals. Coronal views were available in seven subjects years. Histopathological investigation of the excised tissue and revealed an upper vermis split in all seven cases. The revealed a single fibroma without evidence of malignancy. interpeduncular fossa had a normal appearance. The fourth There are no additional clinical signs pointing toward ventricle had a normal shape and, on midsagittal view, the Gorlin–Goltz syndrome (OMIM 109400) in this patient. fastigium was not cranially displaced. Supratentorial anoma- lies were not discernible (Fig. 1). Of note, vermis folia Family 4 dysplasia and upper vermis split are typical findings in Individual II.1 presented with early-onset COMA. The boy Joubert syndrome.14 showed mild motor developmental delay, muscular hypoto- nia, and early-onset ataxia. His speech development is Family 1 currently normal and his cognitive development seems to be Family 1 is a multigenerational family with several individuals normal, but assessment is unsecure at age of only 2 years. showing clinical features of OMA. Individual II.8 was Clinical evaluation of his parents did not reveal any sign diagnosed with early-onset OMA at the age of five months. of OMA. During childhood, ataxia, global developmental delay, and learning disability became apparent. His maternal half-sister, Family 5 individual II.3, had delayed motor and speech development. Individual II.1 presented with early-onset OMA, ataxia, and She attended a special school due to learning disability. She motor as well as speech development delay. As his reports no head jerks or problems with horizontal pursuit, neuroimaging features showed overlap with patterns observed and her relative recalled no signs of OMA during her in other patients of this study, direct testing of SUFU was childhood. Her son (III.3) was diagnosed with OMA at the initiated (Fig. 1i–l). His father, individual I.1, had a age of five months. He presented with ataxia, delay in motor longstanding history of ophthalmological treatment including and speech development as well as cognitive impairment. several surgical interventions for strabismus. His mother used Individual II.5, the sister of II.3, had normal motor, speech, to urge him to practice horizontal gaze pursuit, and he is and cognitive development. Early OMA is not recalled by accustomed to his jerky head movements. Currently, he shows other, older family members. She attended regular school, is mild impairment of horizontal saccades and occasional head now a housewife and mother, and has no vocational training. jerks. A diagnosis of OMA was not established previously, Her son, individual III.6, presented with early-onset OMA, however, and onset of OMA cannot be determined. His ataxia, motor and speech developmental delay as well as cognitive development was normal and he accomplished an cognitive impairment. Clinical data for individual I.2, the academic career. mother of II.3, II.5, and II.8, was limited, but delay in speech development was reported during early childhood. Family 6 Individual II.1 and his younger brother II.2 were both Family 2 diagnosed with early-onset OMA at the age of 6 and Individual II.1 was diagnosed with early-onset OMA, ataxia, 10 months, respectively. Both had early-onset ataxia, and delay in motor and speech development (Fig. 1e–h). His muscular hypotonia, and mild motor as well as speech cognitive performance is normal. His father, individual I.1, developmental delay. Neuroimaging features of individual II.1 was allegedly healthy. However, clinical re-evaluation and resembled the pattern observed in other patients in this study. consultation of the paternal grandmother revealed motor A heterozygous SUFU variant was identified using ES. Clinical developmental delay with impaired balance in his first years history of their mother, individual I.2, revealed unaided of life. He walked without support at two years three months walking at two years of age, mainly due to sickle feet. Besides and had poor motor coordination throughout kindergarten this, her motor and cognitive development was normal and age. Abnormal eye movements or head jerks were not noted. OMA was not reported in childhood nor present in He learned “very late” to ride a bicycle and tie his shoes. At adulthood. school, he easily learned reading, but was not able to write properly. Over the years, these symptoms ameliorated and he Identification of heterozygous truncating SUFU variants was unimpaired as an adult. To identify the underlying genetic cause of COMA, we performed ES of affected individuals from families 1, 2, 3, 4, Family 3 and 6 and their parents. ES data were filtered for de novo, Individual II.1 had early-onset OMA, but normal motor, homozygous, or compound heterozygous variants with a speech, and cognitive development. Mild balance problems coverage of more than six reads, a minimum quality score of were observed in her first years of life, but overt ataxia was not 10, an allele frequency ≥25%, and an minor allele frequency reported in repeated neurological examinations over the years (MAF) <0.5% in the gnomAD database.15 Based on the (Fig. 1a–d). She presented with secondary macrocephaly with similar clinical presentation, we filtered for variants in a head circumference of +2.6 SD at last follow-up at age nine overlapping genes in all affected individuals in these families, years, while the head circumferences of both parents are and we were able to identify variants in only a single

GENETICS in MEDICINE | Volume 0 | Number 0 | Month 5 ARTICLE SCHRÖDER et al overlapping gene in all five families. All affected individuals SAG led to a further increase in target gene expression in both carried heterozygous truncating variants in the SUFU gene wild-type and patient-derived fibroblasts, with higher expres- (Fig. 2). sion levels in patient-derived cells (Fig. S1). Similarly, In individuals II.1 (family 3) and II.1 (family 4) we inhibition of HH signaling using the Smoothened inhibitor identified the heterozygous truncating variants c.479delA (p. vismodegib downregulates HH signaling and target gene His160Leufs*20) and c.1220_1221insT (p.Phe408Valfs*13) in expression to respective basal levels, still resulting in higher SUFU, respectively, and we confirmed the de novo status of activity in vismodegib-treated patient fibroblasts compared these variants in the affected individuals. Individuals III.3 with wild-type fibroblasts (Fig. S1). Nevertheless, the extent of (family 1), II.1 (family 2), and II.1 and II.2 (family 6) inherited HH signaling regulation upon SAG and/or vismodegib SUFU variants from one of their parents, and clinical re- treatment did not differ between COMA patient–derived evaluation revealed mild to moderate clinical symptoms in and control fibroblasts (Fig. S1). Overall, these results suggest individual II.3 (family 1), I.1 (family 2), as well as I.2 (family that the identified truncation variants in SUFU lead to higher 6). In individual II.1 (family 5), we analyzed all exons and basal HH signaling activity, which, however, can be regulated exon–intron boundaries in SUFU by Sanger sequencing in a by exogenous activator or inhibitor treatment, indicating an candidate gene approach based on the characteristic MRI impaired endogenous repression of HH signaling and thus a features. We identified a heterozygous frameshift variant, compromised inhibition of HH target gene expression. c.309_310delAG (p.Arg103Serfs*3), in SUFU that was inherited by the affected father. DISCUSSION All six variants were not observed in any current database of In this study aimed at identification of the gene associated human genetic variations including gnomAD (access date 26 with COMA in patients who show no conclusive neuroima- April 2020), and they are predicted to lead to SUFU protein ging features at initial evaluation and, in particular, do not truncation (Fig. 3). SUFU is a highly conserved protein that is fulfill diagnostic criteria of Joubert syndrome (definite molar under strict mutational constraint. In gnomAD, which tooth sign), we detected heterozygous variants of SUFU in contains the genetic data of more than 250,000 alleles, only 15 subjects from six unrelated families of various ethnic 194 missense variants were observed while 286 were expected backgrounds. Early-onset (congenital) ocular apraxia was well for SUFU, resulting in a z-score of 1.93. Moreover, no documented in ten patients. Eight of them showed additional homozygous truncating variants were observed in SUFU and neurological features including early-onset ataxia and devel- only three heterozygous truncating variants were detected in opmental delay, with some phenotypic variability. Among the gnomAD (probability of loss of function intolerance [pLI] parents carrying familial SUFU variants, two had no history of score of 1), indicating that SUFU is extremely intolerant to OMA or other neurological features (II.5 family 1, I.2 family loss-of-function variants. 6), one had motor and speech developmental delay as well as motor incoordination in his first years of life that regressed SUFU variants lead to impaired repression of HH signaling completely until adult age (I.1 family 2), one showed speech signature genes developmental delay during early childhood (I.2, family 1), SUFU is a negative regulator of the HH signaling pathway. In and one shows mild OMA at adult age with unknown onset the absence of active HH signaling, SUFU binds to cytosolic (I.1, family 5). GLI proteins, thereby restricting their activity and inducing Of note, the neuroimaging features consistently comprise their truncation, which in turn promotes GLI repressor abnormalities of the superior cerebellar peduncles and the formation and induces repression of their target gene upper cerebellar vermis, but no full-blown molar tooth sign as expression. Since the existence of intact primary cilia is seen in typical Joubert syndrome. The molar tooth, now required for HH signaling in vertebrates, we first analyzed considered pathognomonic for Joubert syndrome, arises from whether patient-derived fibroblasts form acetylated tubulin the combination of elongated, thickened, and horizontally positive cilia. However, we did not observe any differences in oriented superior cerebellar peduncles, hypo-/dysplasia of the cilia occurrence, morphology, or ciliary localization of SMO in cerebellar vermis with rostral shifting of the fastigium, and an patient-derived fibroblasts compared with wild-type cells, abnormally deep interpeduncular fossa at the section of the indicating that the identified truncating variants in SUFU brainstem isthmus and upper pons.16 However, it is worth have no impact on this organelle essential for HH signaling mentioning that a milder variant of the molar tooth sign was (Fig. 4a). To assess whether the SUFU variants have an effect occasionally observed in patients carrying variants in certain on the repressor function of SUFU, we analyzed the expression Joubert genes including NPHP1, C5orf42, SUFU, and – of HH signaling target genes in wild-type and COMA FAM149B1.17 20 These observations may suggest that the patient–derived dermal fibroblasts by quantitative real-time neuroimaging features in certain forms of COMA and in PCR (Fig. 4b and S1). Interestingly, we observed a significant Joubert syndrome constitute a continuous spectrum. From increase in the general activity in COMA patient–derived our experience, a clear distinction between a definite molar fibroblasts compared with control cells resulting in higher tooth sign and a milder hindbrain malformation as described basal expression levels of GLI1, GLI2, GLI3, and PTCH1 here is occasionally challenging, even with a technically (Fig. 4b). Treatment of cells with the HH signaling agonist optimal MRI investigation.

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a Family 1 Family 2

12 3 1 2 I WT/Mut WT/Mut WT/WT 1 2 3 4 5 67 8 1 II WT/WT WT/Mut WT/Mut WT/WT WT/Mut WT/Mut 12345678 III WT/Mut WT/Mut

Family 3 Family 4 Family 5 Family 6

11221 21 2 I WT/WT WT/WT WT/WT WT/WTWT/Mut WT/WT WT/WT WT/Mut 1 1 1 2 II WT/Mut WT/Mut WT/Mut WT/Mut WT/Mut

b F1: c.83CT; p.Glu367* F3: c.479delA; p.His160Leufs*20 X X L C P G A FASLFP RQLE SVH SG DHV S W

WT/Mut

WT/WT

F4: c.1220__1221insT; F5: c.309__310delIAG; F6: c.1333dupG; p.Phe408Valfs*13 p.Arg103Serfs*3 p.Glu445Glyfs*22 VCLH SPX GGFR AITFVST GDNRVHE KMLE DLE intron

WT/Mut

WT/WT

Fig. 2 Pedigrees and genetic characterization of six families with congenital ocular motor apraxia carrying heterozygous loss-of-function variants in SUFU. (a) Pedigrees of families 1–6 showing segregation of rare deleterious SUFU variants. Unfilled shapes denote healthy, filled shapes affected individuals. (b) Chromatograms of the identified SUFU variants in family 1 (F1: c.83C>A; p.Ser28*), family 2 (F2: c.1099G>T; p.Glu367*), family 3 (F3: c.479delA; p.His160Leufs*20), family 4 (F4: c.1220_1221insT; p.Phe408Valfs*13), family 5 (F5: c.309_310delAG; p.Arg103Serfs*3), and family 6 (F6: c.1333dupG; p.Glu445Glyfs*22) compared with wild-type (WT) sequences of the respective positions. Localization of frameshift or nonsense variants is indicated in red.

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E1 E2 E3 E4-E6 E7 E8 E9 E10 E11 E12 SUFU

c.83C>A c.479delA c.1220__122insT p.Ser28* p.His160Leufs*20 p.Phe408Valfs*13 c.309__310delAG c.1099G>T c.1333dupG p.Arg103Serfs*3 p.Glu367* p.Glu445GIyfs*22

SUFU SUFU SUFU__C

p.Gly19Val p.His176Arg p.Arg299* c.1022+1G>A p.IIe406Thr p.Ser481Asn p.Ile37Val c.598-1G>C p.Glu283Argfs*3 p.(Ser399Tyrfs*4) p.Lys442Arg p.Asn319Thrfs*42 c.1365+2T>A

Fig. 3 Overview of the identified heterozygous loss-of-function variants in SUFU on genomic and protein level. Schematic diagram of the SUFU gene (top panel) and protein (bottom panel) showing the localization of six truncating variants identified within this study in affected individuals from six independent families (black). Localization of SUFU variants associated in previous studies with Joubert syndrome are indicated in green and blue; variants associated with Gorlin–Goltz syndrome are marked in red.19,22,27,28.

a control b fibroblasts COMA-patient derived fibroblasts 100 100

10 10 GLI1/18S 1 GLI1/HPRT 1 Smo -fold -fold -fold -fold

0.1 0.1 controls COMA controls COMA

10 10

1 1 GLI2/18S 0.1 GLI2/HPRT 0.1 -fold -fold -fold -fold

acetyl. tubulin acetyl. 0.01 0.01 controls COMA controls COMA

10 10

1 1 GLI3/18S GLI3/HPRT Smo -fold -fold -fold -fold acetyl. tubulin acetyl. 0.1 0.1 controls COMA controls COMA

10 10

1 1 PTCH/18S Smo PTCH/HPRT DAPI -fold -fold -fold -fold acetyl. tubulin acetyl. 0.1 0.1 controls COMA controls COMA

Fig. 4 Cilia formation and expression of Hedgehog signaling signature genes in congenital ocular motor apraxia (COMA) patient–derived dermal fibroblasts. (a) Representative pictures of double immunofluorescent staining for visualization of SMO (upper row in monochrome, third and lower row in red) and acetylated tubulin (acetyl.tubulin) (second row in monochrome, third and lower row in green) in control fibroblasts and fibroblasts derived from affected individual II.2 (family 3, first column), individual II.1 (family 2, middle column), and individual II.1 (family 4, right column). Nuclei were visualized by DAPI staining (lower row in blue). Scale bars: 1 µm. (b) Quantitative real-time polymerase chain reaction (PCR)–based expression analyses of the Hedgehog signaling signature genes GLI1, GLI2, GLI3, and PTCH1 normalized to 18S ribosomal RNA (rRNA) (left column) or HPRT (right column) expression levels, respectively, of controls (N = 5) and COMA patient–derived fibroblasts (COMA) (N = 4). Shown results represent data of two different cellular passages per fibroblast culture each analyzed in biological triplicates (gray circles) that were measured in technical triplicates. Total mean values +/- SEM of all analyzed samples are indicated in black. Significant differences were tested by nonparametric Mann–Whitney tests. *p < 0.05; **p < 0.01; ***p < 0.001. No significantly different expression levels were observed in the five independent control fibroblast cultures, different cellular passages, or biological triplicates. For comparison of individual gene expression levels of the four independent COMA patient–derived fibroblast cultures as well as HIP expression levels see Supplemental Data (Fig. S1).

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A separation of SUFU-associated COMA as reported here fibromas, and ocular anomalies can be observed in patients and Joubert syndrome based solely on clinical criteria is even with BCNS.24 Among the 11 subjects with heterozygous more difficult and will not be possible in many cases. The SUFU variants reported here, two showed macrocephaly and clinical spectrum of Joubert syndrome encompasses a wide one had borderline head circumference (+2.0 SD; I.1 family range of phenotypes spanning from mild variants with 2). Subject II.1 from family 3 carrying the de novo muscular hypotonia, ataxia, OMA, and benign developmental heterozygous SUFU variant c.479delA shows secondary, delay to severe forms with pronounced, sometimes progres- nonfamilial macrocephaly as a feature consistent with BCNS. sive, multisystem disorder.14,16 Thus, there is considerable At age six years a single digital fibroma was excised. In BCNS, phenotypic overlap between these conditions. ovarian and cardiac fibromas are minor diagnostic criteria, The SUFU variants that we detected in the subjects but digital fibromas were not reported, to our knowledge. described here were exclusively truncating variants. These Subject I.1 from family 5 shows macrocephaly (+3.0 SD) and variants are distributed over the entire protein (Fig. 3), thus tall stature (body height 195 cm, +2.1 SD). Parental head making haploinsufficiency the most likely underlying patho- circumferences were not available. physiological mechanism. On a cellular level, these variants Besides this, we found no other features consistent with seem not to impair the occurrence, morphology or SMO BCNS based on physical examination and ultrasound. Thus, localization of the primary . SUFU is a major inhibitor the condition reported here and SUFU-associated BCNS are of HH signaling, which is an evolutionary highly conserved likely allelic disorders, as there is currently no convincing pathway that plays an important role in embryonic develop- evidence for a clinical overlap. Both, somatic and germline ment, stem cell maintenance, and tissue homeostasis. (heterozygous and biallelic) variants in SUFU were found to In knockout mice with SuFu deficiency targeted to the be associated with cerebellar medulloblastoma.25 A hetero- cerebellum and some parts of the midbrain, it was demon- zygous truncating germline variant in SUFU was observed in strated that SuFu is required for proper midhindbrain familial meningioma.26 Given the fact that haploinsuffi- patterning, controls cerebellar patterning by regulating cell ciency caused by truncating loss-of-function variants differentiation and migration, and regulates the localization throughout SUFU is the underlying molecular mechanism and level of SHH signaling and the levels of GLIs, GLI3R in in both COMA and BCNS, additional genetic and/or particular, and that GLI3R partially mediates SuFu functions nongenetic modifiers must exist that drive the phenotypic during cerebellar morphogenesis.21 This prompted us to expression toward a specific clinical entity. We did not analyze the mutational impact on this pathway. Interestingly, observe any clusters of variants in SUFU or other pathway we observed a higher basal activity of HH signaling in COMA genes in our exome data, which might point toward a patient–derived fibroblasts compared with control cells, genetic modifier. However, thenumberofpatientsavailable supporting the hypothesis that the inhibitory function of for this analysis was limited. Consequently, larger cohorts of SUFU is impaired by the identified SUFU variants. SAG- patients with COMA and BCNS are needed in future studies induced stimulation of HH signaling led to a further increase to identify these modifying factors that are determining the in target gene expression levels in both wild-type and patient- phenotypic outcome of heterozygous loss-of-function var- derived fibroblasts, whereas inhibition of HH signaling by the iants in SUFU. Smoothened inhibitor vismodegib reduced HH signaling and More recently, germline biallelic pathogenic variants of target gene expression to the respective basal levels. This SUFU were shown to impair SHH signaling and to be shows that although basal activity of the HH signaling associated with Joubert syndrome type 32 (OMIM 617757).19 pathway is higher in COMA patient cells, pathway regulation Homozygous missense variants in SUFU were detected in four is not impaired. Remarkably, COMA patient–derived fibro- children from two unrelated families. All subjects showed blasts show a similar fold change of HH pathway activation facial dysmorphism including hypertelorism; broad, and repression as wild-type cells, if values were normalized to depressed nasal bridge; and frontal bossing, as well as the respective basal levels. developmental delay with mild intellectual impairment, gait Interestingly, germline heterozygous truncating and loss-of- ataxia, and dysarthria. Three of them had postaxial poly- function variants in SUFU (OMIM *607035) were observed in dactyly and two had global macrosomia with macrocephaly. the basal cell nevus syndrome (BCNS, Gorlin–Goltz syn- Neuroimaging revealed cerebellar vermis hypoplasia with drome, OMIM 109400), a cancer-predisposing condition with elongated superior cerebellar peduncles, designated by the variable developmental and skeletal anomalies.22,23 BCNS authors as a “mild molar tooth sign.” Furthermore, poly- may also be associated with a heterozygous germline microgyria was present in two siblings.19 Interestingly, all four pathogenic variant in PTCH1. BCNS is mainly characterized patients presented with ocular motor apraxia, whereas their by lamellar calcification of the falx, jaw , palmar/ parents, who were heterozygotes of one of the identified plantar pits, and multiple basal cell carcinomas as major missense variants in SUFU, did not show any signs of Joubert diagnostic criteria. Additionally, childhood medulloblastoma, syndrome or COMA, suggesting that heterozygous truncating lymphomesenteric or pleural cysts, macrocephaly, cleft lip/ variants have a more severe effect on SUFU function as a palate, vertebral/rib anomalies (bifid/splayed/extra ribs; bifid repressor of the SHH pathway activity than heterozygous vertebrae), preaxial or postaxial polydactyly, ovarian/cardiac hypomorphic missense variants which were identified in

GENETICS in MEDICINE | Volume 0 | Number 0 | Month 9 ARTICLE SCHRÖDER et al clinically unremarkable parents of patients with Joubert assistance, and Karin Boss for critically reading the manuscript. syndrome by De Mori et al.19 We thank Karl Kugler (Munich, Germany) for providing clinical Besides these two biallelic missense variants, De Mori data and Christina Mitteldorf (Department of Dermatology, et al.19 additionally identified four individuals with hetero- Venereology and Allergology, University Medical Center Göttin- zygous, truncating variants in SUFU, c.598-1G>C, c.846dupC, gen, Germany) for expert investigation of the digital fibroma of c.895C>T, and c.1192_1193delAA, which were either inher- subject I.1 from family 3. This work was supported by the ited from a parent or occurred de novo (Fig. 3). No details on Niedersächsisches Ministerium für Wissenschaft und Kultur by the clinical presentation of these subjects were provided, grant 74ZN1284 to K.B., the Deutsche Forschungsgemeinschaft especially not regarding the severity of their phenotype and (DFG, German Research Foundation) under Research Group FOR the presence of a molar tooth sign, but in line with the 2800 “Chromosome Instability: Cross-talk of DNA replication individuals presented in our study, De Mori et al.19 observed stress and mitotic dysfunction” and Germany’s Excellence no tumors or any signs of Gorlin syndrome in their subjects. Strategy, Cluster of Excellence “Multiscale Bioimaging: from Parents inheriting these variants were indicated as healthy. Molecular Machines to Networks of Excitable Cells” (MBExC) Still, based on the results of our study we observed a mild to B.W. neurodevelopmental phenotype during early childhood in three parents including delayed motor and speech develop- FUNDING ment without signs of OMA (individual II.3, family 1), Open Access funding enabled and organized by Projekt DEAL. impaired balance and poor motor coordination (individual I.1, family 2), and ongoing ophthalmological treatment from DISCLOSURE early childhood on (individual I.1, family 5). Clinical re- The authors declare no conflicts of interest. evaluation of the subjects with heterozygous truncating 19 variants identified by De Mori et al. might help to determine Publisher’s note Springer Nature remains neutral with regard to clinical significance of these variants and evaluate the clinical jurisdictional claims in published maps and institutional spectrum of SUFU-associated variants. affiliations. 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